After the concept of specific folic acid binding protein (FBP) had been brought forward (Johns et al.: J. Clin. Invest. 1961; 40: 1684), Rothernberg et al., first found FBP in cells of human body (Proc. Soc. Expl. Biol. Med. 1970; 133: 428. J. Clin. Invest. 1971; 50: 717), and Lesli et al., also isolated FBP from cell membranes (Biochem. 1972; 11: 1969). Antony et al. systematically studied on placenta cells and definitely proposed that said FBP has a function as folic acid receptor (FR) on the cell membrane (J Biol Chem 1981; 256(18): 9684), and systematically and successively illustrated the biochemical properties of FR (Blood 1992; 79 (11): 2807, Annu. Rev. Nutr. 1996; 16: 501) FR is a kind of FBP anchored on glycerophosphatide inositol (GPI) in cell membranes, which can be cut from the cell membrane by GPI-specific phosphatidase C or D (Lee et al: Biochem. 1992: 31: 3236, Verria et a: J Biol. Chem. 1992; 267(6): 4119). FR is uniformly located on the surface of cell membrane essentially, and after binding with folic acid, it can be shifted into coated pits or coveolae, upon promoted by the initiator, and therefore be clustered (Mayor et al: Science 1994; 264: 1948), then folic acid is transferred into the cell by the resulted endocytosis (Anderson et al: Science 1992,
There are mainly three forms of FR in human body: FR-α FR-β and FR-γ, wherein FR-γ is a kind of secreting protein expressed on hematopoietic cells (Shen et al: Biochem 1995; 34: 5660). FR-α and FR-β also exist in the surface of animal cell, wherein FR-α is mainly expressed on tumor cells and kidney cells, and FR-β is expressed on hepatic cells. Folic acid can adjust the affinity and density of cells to a certain extent. After limiting the taking dosage of folic acid, the affinity of FR-α to folic acid decreases, the density of FR-α increases in tumor cell and decreases in kidney cell, while that of FR-β is not obviously affected (Gates et al: Clin Cancer Res 1996; 2:1135).
With the disclosures that the expression number or activities of FR on most tumor cells are remarkably higher than that on normal cells (Cambell et al: Cancer Res 1991; 51: 5329. Coney et al: Cancer Res 1991; 51:6125. Weitman et al: Cancer Res 1992; 52:3396), the researches of using folic acid as guiding media to target tumor cells develop rapidly.
The results of animal experiments of tumor image diagnosis show that the complexs of folic acid as FR ligand directly or indirectly binding with radionuclides have remarkable target effect on tumor site (Low et al: WO96/36367 Nov. 21, 1996; U.S. Pat. No. 5,688,488 Nov. 18, 1997).
The cell culturing results of folic acid-PEG-liposome obtained by indirectly binding folic acid with surface of liposome show that the target effect to tumor cells of said folic acid-PEG-liposome is better than that of PEG-liposome or normal liposome (Lee et al: J Biol Chem 1994; 269(5), 3198. Wang et al: Proc Natl Acad Sci USA 1995; 92:3318. Lee et al: Biochim Biophys Acta 1995: 1233:134. Vogel et al: J Am Chem Soc 1996; 118(7): 1581. Thompson et al: WO97/31624 Sep. 4, 1997. Lu, Yaowei et al: Transaction of Shanghai University of Medical Science 2000; 27(1): 4).
The complexs of folic acid-polymer can transfer and release wholly said polymer into non-lysosome plasma of cells via FR. The bovine serum albumin, bovine immune albumin, horseradish peroxidase, ribonuclease, bean seninase inhibitor and anti-DNA oligonucleotide, which are bound with folic acid, can obviously be introduced into KB cells (human nasopharyngeal carcinoma cells), Hela cells (human cervical carcinoma cells) and XC cells (fibroblasts transfected with Rous sarcoma virus) to show their corresponding effects (Leamon et al Proc Natl Acad Sci USA 1991, 88:5572. Low et al: WO90/12096 Oct. 18, 1990). The anti-T cell receptor monoclonal antibody or anti-Fc receptor monoclonal antibody, which are bound with folio acid, can closely bind tumor cell, T-cell or natural killer cell, monocytes and macrophages together to fulfill the purpose of resolving said tumor cell (David et al: WO96/34630 Nov. 7, 1996). Moreover, after bound with folic acid, the toxins (Momordin, a kind of protein toxin its cytotoxicity can be shown only after passing through ribosome and entering into cell plasma, exotoxin fragments of pseudomonad (LysPE38 and CysPE35)) having function of inhibiting synthesis of protein show greatly improved abilities to suppress the growth of tumor cells (Leamon et al: j Biol Chem 1992; 267(35); 249666. 1993: 268(33): 24847).
Dextran (dextran) is a polymer of D-glucose obtained by fermenting saccharose with leuconastoc mesenteroidas (Gronwall et al: Acta Physiol Scand 1994; 7:97. 1945; 9:1. U.S. Pat. No. 2,437,518. U.S. Pat. No. 2,644,815). The linking manners of glucosyls in Dextran are different between each other of different Dextranes obtained with different strains, but the main linking manner is α-1,6 bond, and the others are α-1,4 bond or α-1,3 bond (Van Cleve et al: J Am Chem Soc 1956; 78:4435. Xu, Danfeng et al: Transaction of Pharmacology 1986; 21(3), 204). The animal experiments and tracing results of clinic experiments show that, after injecting Dextran, no abnormity and tissue damage is found in substantive organs of animals (Boyd et al: Lancet 1953; 1: 59. Gronwall et al: Acta Physiol Scand 1945; 9: 1), and no accumulation of Dextran exist in liver, spleen, kidney, lung and other organs of human body (Wilkinson et al: J Interal chir 1951; 11: 186). Clinically, Dextran is mainly used as blood volume enlarging agent (Gelin et al: Acta Chir Scand 1961; 122: 309) and blood fluidity improving agent (Gelin: Sock Pathogenesis and Therapy 1962; P332) to treat hemorrhagic shock, burn and liver-kidney syndrome, acute thrombus, thrombotic occlusive angiitis, cardiac infarction, generalized sclerosis and etc.
Because Dextran has specific biologic feature of having multiple hydroxyl groups, it has been used as carrier for many medicaments to fulfill the purposes of strengthening chemical stability of said medicaments or improving biologic availability of said medicaments or diagnosing diseases of lymphatic system. These Dextran-medicament complexs includes: Dextran-antimony (Mikhail et al: Exptl Parasitol 1975; 37: 348), Dextran-iron (Beresford et al: Brit J Pharmacol 1957; 12: 107), Dextran-insulin (Armstrong et al: Biochim Biophys Res Comm 1972; 47: 354), Dextran-daunomycin (Bernsten et al: J Nalt Cancer Inst 1978; 60(2): 379), Dextran-mitomycin C (Kojima et al: J Pharmacol 1980; 32:30), Dextran-Vitamin B12 (Scrollini en al Eur J Med Chem 1974; 9: 621), Dextran-amethopterin (Hubert et al: EP0383170A2), Dextran-α (or β)-diastase (or trypsase) (Marshall et al: Arch Biochem Biophys 1975; 167:777), Dextran-sulfate (Kozo Yamada et al: Jap Circul j 1961; 25: 570, 575, 579), radioactive technetium (99mTc)-Dextran (Henze et al: J Nucl Med 1982; 23: 923. Ercan et al: Eur J Nucl Med 1985: 11: 80. Lu, Weiyao et al: Transaction of Shanghai University of Medical Science 1991; 18(4): 246. Liu, Yongchang et al: China J Nucl Med 1993; 13(3): 143). Among above Dextran-medicament complexs, the 99mTc-Dextran 105 injection and stannous-Dextran 105 injection, which are developed by the inventor and used for locating affected part of diseases of lymphatic system and for assistant diagnosis of lymphatic metastasis tumor, have been formally produced and clinically used.
In sum, the FR on the surface of tumor cell is an effective pathway to introduce medicaments of radionuclide, liposome and polymer into said tumor cell via folic acid; Dextran is studied for a long time used as blood volume enlarging agent and carrier of radionuclide and other medicament, but the Dextran per se shows no antineoplastic effect. So far, there is no document or patent to research complexs of folic acid and polysaccharides, especially the complexs of folic acid and Dextran, and their uses as antineoplastics.